TECHNICAL BULLETIN. INFORCE 3: Aids in the prevention of respiratory disease caused by infectious bovine rhinotracheitis. INF
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1 INF TECHNICAL BULLETIN January 2016 INFORCE 3: Aids in the prevention of respiratory disease caused by infectious bovine rhinotracheitis. Zoetis 100 Campus Drive Florham Park, New Jersey SUMMARY Two vaccination/challenge efficacy studies were conducted to assess the ability of INFORCE 3 to help protect cattle from respiratory disease caused by infectious bovine rhinotracheitis (IBR) virus when administered as a single intranasal dose. 1,2 Animals in both studies were challenged intranasally by instillation of a 2 ml dose containing the virulent IBR virus (Cooper strain). In Study 1, using 7- to 9-month-old calves, INFORCE 3 vaccinates had: Significantly lower incidence of clinical disease (morbidity* = 5% vs. 100%) (P ) Significantly less duration of disease (percent days ill = 0.07% vs %) (P ) Significantly lower rectal temperatures 1 day and 3 to 9 days following challenge (P ) Significantly less virus shedding on Days 5 through 11 post-challenge (P ) In Study 2, using 3- to 8-day-old calves, INFORCE 3 vaccinates had: Significantly lower incidence of clinical disease (morbidity* = 5.6% vs. 100%) (P ) Significantly less duration of disease (percent days ill = 0.02% vs %) (P ) Significantly less virus shedding on Days 2 through 13 post-challenge (P ) Based on the significant protection demonstrated in Study 1, the USDA granted INFORCE 3 the label claim aids in the prevention of respiratory disease caused by IBR. *Morbidity was defined as whether an animal ever exhibited acute clinical IBR disease (positive morbidity score) following challenge. INFORCE 3 aids in the prevention of respiratory disease caused by IBR and PI 3, and prevents BRSV respiratory disease. In addition, extensive safety studies were conducted with INFORCE 3, demonstrating safety in all ages and classes of animals, including newborn calves and high-stress stockers.
2 Minimum Immunizing Dose Minimum immunizing dose (MID) levels are established prior to licensing of a vaccine. Expiration levels of an MLV product are set at 0.7 log higher than MID, therefore, by using MID levels for vaccine antigens, investigators put the vaccine at its maximum potential disadvantage at the time of challenge. When a vaccine withstands challenge under these circumstances, it will be at least as effective when the antigen level is at its release level. Overview of the IBR respiratory efficacy studies Study 1 animals Healthy weaned calves were selected for this trial. The calves were 7 to 9 months of age at the time of vaccination and were sero-negative (serum neutralizing [SN] antibody titer of <1:2) for IBR virus and not persistently infected (PI) with BVDV. Study 2 animals Healthy colostrum-deprived bull calves were selected for this trial. The calves were 3 to 8 days of age at the time of vaccination and were seronegative for IBR virus and not PI with BVDV. Study Design Both studies were divided into two phases: the vaccination phase and the challenge phase as described in Tables 1 and 2. For both studies, all animals were vaccinated on Day 0, challenged on Day 28 and monitored for virus isolation, rectal temperatures and clinical signs of IBR disease for 14 days post-challenge. Table 1 Study 1 design Vaccine Placebo Control (BVD1-BVD2-PI 3 -BRSV) INFORCE 3 (IBR-BVD1-BVD2-PI 3 -BRSV) 1 1 ml per naris N Test vaccine For both studies, the placebo control calves did not receive IBR. The placebo control animals received either a vaccine that contained BVD, PI 3 and BRSV (Study 1) or PI 3 and BRSV (Study 2) at levels above the minimum immunizing dose (MID) for each fraction. The calves vaccinated with INFORCE 3 (T02) were administered a vaccine that had the MID level of the IBR virus and the remaining fractions (BVD, PI 3 and BRSV for Study 1) or (PI3 and BRSV for Study 2) at or above their respective MIDs. Vaccination phase For both studies, on Days 0 through to 2 days before challenge, all animals were observed once daily for general health. For both studies, on Day 0, calves were vaccinated intranasally with 2 ml (1 ml per naris), with the appropriate vaccine as described in Tables 1 and 2. Animals were observed for any local and/or systemic reactions associated with vaccination (depression, trembling, tachypnea) once within four hours following vaccination. No Vaccination Phase Challenge Phase (Days) Clinical Day Dose Route 1 Challenge Observations ml IN ml IN Table 2 Study 2 design Vaccine Placebo Control (PI 3 -BRSV) INFORCE 3 (IBR-PI 3 -BRSV) N Vaccination Phase Challenge Phase (Days) Clinical Day Dose Route Challenge Observations ml IN ml IN ml per naris One animal removed from study on Day 28 prior to challenge due to injury One animal removed from study on Day 40 post-challenge due to illness not related to IBR disease 2
3 reactions were noted due to vaccinations. For Study 1: Animals were randomly assigned by treatment group to three rooms for the vaccination phase. During the challenge phase, animals were commingled and randomly assigned into three identical isolation rooms. Two of the three rooms contained three controls and six vaccinates, and the third room contained four controls and eight vaccinates. For Study 2: Animals were housed in a biosafety level-2 facility for the duration of the study. During the vaccination phase, animals were housed in identical individual pens contained in two separate but identical rooms by treatment group. During the challenge phase, animals were randomly assigned to individual pens in two rooms so that both treatment groups were represented in each room. Samples and assay of specimens For Study 1, blood samples were collected from all calves on Days 0 (prevaccination), 14, 28 (pre-challenge) and 42. The serum was tested for SN antibodies to IBR. Nasal secretions were collected using swabs on Days 27 and 29 through 42 from each animal for virus isolation (VI). All serologic and VI assays were completed at Zoetis Laboratory Sciences in Kalamazoo, Mich. For Study 2, blood samples were collected from all calves on Days 0 (prevaccination), 27 (pre-challenge) and 42. The serum was tested for SN antibodies to IBR. Nasal secretions were collected using swabs on Days 27 through 42 (excluding Day 28) from each animal for VI. All serologic and VI assays were completed at Zoetis Laboratory Sciences in Kalamazoo, Mich. Challenge phase (procedure and observations) For both studies, animals were challenged intranasally by instillation of a 4 ml dose (approximately 2 ml per nostril) of virulent IBR (Cooper strain) using a compressed air nebulizer. From three days prior to challenge through 14 days post-challenge, all calves were monitored for clinical signs of IBR respiratory disease and daily rectal temperatures were recorded. Challenge virus For Study 1, the IBR challenge virus was prepared at the Zoetis facility in Lincoln, Neb., as per United States Department of Agriculture, Animal and Plant Health Inspection Services, Center for Veterinary Biologics (USDA-APHIS-CVB) instructions. For Study 2, the IBR challenge virus was prepared at Zoetis Veterinary Medicine Research and Development (VMRD), Kalamazoo, Mich., as per USDA-APHIS- CVB instructions. Masking Post-challenge clinical monitoring, clinical sampling and laboratory testing were conducted and recorded without the knowledge of treatment group assignment. Results Study 1 Serology On Days 0, 14 and 28, all placebo control calves were sero-negative for IBR SN Table 3 Geometric means and geometric least squares means (LSMs)* of IBR-specific SN antibody titers (percent seroconversion rate) Treatment Study Days Treatment Group T01 Placebo Control 1 (0) 1 (0) 1.0 a (0) 44.5 a (100.0) T02 INFORCE 3 1 (0) 1.1 (5.0) 1.3 a (10) 62.4 a (100.0) Days 0 and 14 are geometric means and Days 28 and 42 are geometric LSMs* P 0.05). *Since animals were housed separately by treatment group during the Day 0 and 14 time-points of this study, only descriptive geometric means can be calculated for treatment groups during that time period. (No statistical analysis is appropriate since treatment is completely confounded with housing.) Once animals from the treatment groups were commingled (Day 28 and Day 42), analysis was possible and therefore we could calculate geometric LSMs (An LSM (Least Squares Mean) indicates that these are estimates from a statistical model). 3
4 antibodies (<1:2). Vaccine induced detectable IBR SN antibody responses ( 1:2) were observed in two of 20 (10%) vaccinates on Day 28. Following challenge, all animals in both groups seroconverted. Although the vaccine induced limited seroconversion, it primed immunity as demonstrated by development of strong anamnestic SN antibody responses postchallenge (Table 3). Intranasal vaccination does not induce an appreciable systemic antibody response to IBR vaccine and hence SN antibodies should not be used as a method of determining response or exposure to vaccination. Figure 1: LSM rectal temperatures before and after IBR challenge Rectal temperatures on Days 26, 27 and 28 are expressed as geometric means Least Squares Mean Virus Titers Log 10 TCID 50 /ml Figure 2: IBR virus isolation titers after challenge Rectal temperatures The post-challenge LSM rectal temperature profiles of vaccinates and controls are shown in Figure 1. The LSM rectal temperatures of vaccinates were significantly lower (P ) than those of the controls on Days 29 and 31 through 37. Clinical disease The virulent IBR challenge induced acute clinical disease in all control animals (Table 4). In comparison, only one of 20 (5%) vaccinated animals developed acute IBR clinical disease. The LSM percent days (see box for definition) with disease for the placebo controls was compared with 0.07 for the vaccinates (P ). Virus shedding Virus isolation results are summarized in Figure 2. The control animals shed significantly more virus than vaccinates on Study Days 33 to 39 (P ). Discussion Study 1 The objective of this study was to demonstrate the IBR efficacy of INFORCE 3 in naïve calves. The study was valid because all control animals were sero-negative (serum neutralizing [SN] antibody titer of <1:2) for IBR specific antibodies prior to the start of the study and developed severe disease consisting of fever and acute IBR clinical disease following challenge. Rectal temperatures in vaccinates were significantly lower (P ) than those in the control animals on Days 1 and 3 through 9 following challenge. While all control calves exhibited acute IBR disease following challenge, only one vaccinated calf was clinically ill. The level of IBR in nasal secretions of vaccinates was significantly lower (P ) from 5 days through 11 following challenge compared with controls, indicating a clinically relevant reduction in virus shedding. Vaccination using tsibr by the intranasal route of administration did not give a detectable systemic antibody response and cannot be used as a method of determining response to vaccination. No attempt was made to measure local (upper respiratory) immunity or cell mediated immunity; however, given the level of efficacy observed following challenge, it 4
5 is reasonable to assume that INFORCE 3 induced a protective immune response even though systemic neutralizing antibody was not measurable in the majority of the vaccinates at the time of challenge and the protection induced against challenge. Results Study 2 Serology On Days 0 and 27, all placebo control calves were sero-negative for IBR SN antibodies (<1:2). Vaccine induced detectable IBR SN antibody responses ( 1:2) in eight of 18 (44.4%) vaccinates on Day 27 (pre-challenge). Following challenge, all animals seroconverted (Table 5). Although the IBR systemic neutralizing antibody response to vaccination was poor, upon exposure to the disease, a strong anamnestic response developed. Intranasal vaccination will not give an appreciable systemic antibody response in all animals and therefore SN antibodies should not be used as a method of determining response to vaccination. Rectal temperatures The post-challenge LSM rectal temperature profiles of vaccinates and controls are shown in Figure 3. The LSM rectal temperatures of vaccinates were significantly lower (P ) than those of the controls on Days 31 through 37. Clinical disease The virulent IBR challenge induced acute clinical disease (morbidity) in all control animals (17/17). These calves were morbid for one or more days during the challenge phase of the study (Table 6). In contrast, only one of 18 (5.6%) IBR-vaccinated animals developed acute IBR clinical disease for one day. The LSM percent days with disease for the placebo controls was compared with 0.02 for the vaccinates (P ). Table 4 Frequency of clinical disease following challenge Percentage of Animals that Developed Clinical Study Day Disease (Morbidity) Placebo Control INFORCE Disease if ever present LSM percent Positive Days Morbid (P ) 10 of 10 1 of 20 (100%) a (5%) b a 0.07 b Virus shedding Virus isolation results are summarized in Figure 4. The control animals shed significantly more virus than vaccinates on Study Days 30 to 41 (P ). Table 5 Individual geometric LSM IBR serum neutralizing antibody titers and seroconversion rate (%) Treatment Study Days Treatment Group T01 Placebo Control 1 (0) 1.0 (0) a 13.4 (100.0) a T02 INFORCE 3 1 (0) 1.7 (44.4) b 53.7 (100.0) b P ). Clinical assessments of respiratory disease were made according to the following criteria: No = Normal animal. Yes = Acute clinical IBR present: Clinical signs may include depression (animal tends to lie down frequently, moves and responds to stimuli reluctantly, holds head low, staggers and shows little interest in surroundings), mucopurulent nasal discharge and increased respiratory effort (rapid abdominal breathing, and labored and audible grunts). Understanding Least Squares Mean Percent Days The term Least Squares (LS) Mean Percent Days is used by Zoetis scientists to help characterize the extent that a particular disease parameter was observed in groups of test animals following challenge. LS mean percent days factors in the time intervals that animals are affected as well as the percentage of animals affected. In brief, LS mean percent days is the average percent of days animals in a treatment group were afflicted with a given condition. 5
6 Table 6 Frequency of clinical disease following challenge Percentage of Animals that Developed Clinical Study Day Disease (Morbidity) Placebo Control INFORCE Disease if ever present LSM percent a Positive Days Morbid 17 of 17 (100%) a 1 of 18 (5.6%) b 0.02 b One animal was removed from study prior to challenge due to injury (P ) Discussion Study 2 The objective of this study was to demonstrate the IBR efficacy of INFORCE 3 in naïve 3- to 8-day-old calves. The study was valid because all control calves (18) remained sero-negative until challenge and all (17) had IBR disease following challenge with virulent IBR virus. The LSM rectal temperatures of vaccinates were significantly lower (P ) than those of the controls on Days 31 through 37. While all control calves exhibited acute IBR disease following challenge, only one vaccinated calf was clinically ill. The level of IBR in nasal secretions from the control animals was significantly higher than vaccinated animals on 12 days post-challenge (P ). Consistent with Study 1, vaccination using tsibr by the intranasal route of administration did not give a detectable and appreciable systemic antibody response in all animals and therefore SN antibodies should not be used as a method of determining response to vaccination. No attempt was made to measure local (upper respiratory) immunity or cell mediated immunity; however, given the level of efficacy observed following challenge, it is reasonable to assume that INFORCE 3 induced a strong protective immune response even though systemic neutralizing antibody was not measurable in the majority of the vaccinates at the time of challenge. The vaccinates did appear to be primed to IBR by the strong anamnestic SN antibody response seen following challenge. Least Squares Mean Virus Titers LOG 10 TCID 50 /ml Figure 3: Least squares mean rectal temperatures before and after IBR challenge Figure 4: IBR virus isolation titers after challenge 6
7 Conclusion Study 1 and Study 2 Based on the significant protection demonstrated in Study 1, the USDA granted INFORCE 3 the label claim aids in the prevention of respiratory disease caused by IBR disease. Study 2 data demonstrates that INFORCE 3 is effective when used in 3- to 8-day-old calves, which is important because the immune system of neonatal calves is significantly different from that of older cattle. 7
8 REFERENCES 1. Data on file, Study Report No. 3131R , Zoetis, Inc. 2. Data on file, Study Report No. 3131R , Zoetis, Inc. All trademarks are the property of Zoetis Inc., its affiliates and/or its licensors. All other trademarks are the property of their respective owners Zoetis Inc. All rights reserved. INF
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